13C solid-state NMR study of structural heterogeneity in peptides containing both polyalanine and repeated GGA sequences as a local structural model of Nephila clavipes dragline silk (Spidroin 1)
13C solid-state NMR study of structural heterogeneity in peptides containing both polyalanine and repeated GGA sequences as a local structural model of Nephila clavipes dragline silk (Spidroin 1)
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DOI：
10.1021/ma047660z
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发表时间：
2005-04-19
期刊：
影响因子：
5.5
通讯作者：
Nakazawa, Y
中科院分区：
文献类型：
--
作者：
Asakura, T;Yang, MY;Nakazawa, Y
为了避免在测定天然蜘蛛丝结构时可能出现的歧义，我们制备了一种非标记肽，其序列同时包含聚丙氨酸和重复的GGA区域，QGAG(a)(6)GGAGA(GGA)(3) ggaggglgg (I)，和c -13标记肽QGAGAAA[1- c -13]A(8)AAGG[2-C-13]A(13)GAGGAG[2-C-13]G(20)[3-C-13]A(21) ggagaggglgg （Ia）和QGAGAAAAAAGGAGAG-GAG [1- c -13]G(20)[1- c -13]A(21) ggagaggglgg （II）作为蜘蛛拖丝中蜘蛛蛋白1 （MaSpl）的局部结构模型。在核磁共振测量之前的溶剂处理诱导了这些模型肽的结构变化，并提供了一个模型来重现丝绸纤维的结构。构象相关的C-13核磁共振化学位移主要用于确定局部结构，包括评估几个构象的分数。将Ia和II肽溶解于9 M LiBr中，经水透析后，发现其特征结构为：多Ala区α(8)残基为65%的β -片状结构，α(21)残基为70%的3(1)-螺旋结构，GG(20)A（21）序列中Gly（20）残基主要为3(1)-螺旋结构。经过此处理的肽II的Ala（21）残基的二维自旋扩散13C固态核磁共振谱也通过70%的3(1)-螺旋（phi, rho = -90度，120度）和30%的β -片（phi, rho = -150度，150度）结构再现。然而，Ia光谱中分配给31-helix的Ala C-beta峰很宽，这意味着Ala（21）残基的扭转角是分布的，但其平均值近似对应于31-helix的扭转角。在甲酸中溶解，然后在空气中干燥后，观察到la在poly-Ala和GG(20)A（21）区域中β -sheet的比例增加。此外，Ia在甲酸中溶解后，再在甲醇中沉淀，光谱显示出紧密排列的β -片结构，β -片的比例进一步增加，但仍有15%的3(1)-螺旋留在GG(20) a（21）区。poly-Ala区域的β -sheet结构和重复GGA序列中的3(1)-螺旋结构和β -sheet结构与先前NMR研究中来自N. clavipes的本地蜘蛛拖丝纤维的结构模型一致（van Beek， J. D.; et al.）。Proc。国家的。学会科学。美国，2002,99,10266-10271)。另一方面，经三氟乙酸处理的肽以a-螺旋构象为主，其他构象也有显著贡献。其他结构的比例为20-40%，这取决于c -13标记的Ala残基在链中的位置。
To avoid potential ambiguity in the structural determination of native spider silk, we prepared both a nonlabeled peptide with a sequence containing both the polyalanine and the repeated GGA regions, QGAG(A)(6)GGAGA(GGA)(3)GAGRGGLGG (I), and the C-13-labeled peptides QGAGAAA[1-C-13]A(8)AAGG[2-C-13]A(13)GAGGAG[2-C-13]G(20)[3-C-13]A(21)GGAGAGRGGLGG (Ia) and QGAGAAAAAAGGAGAG-GAG [1-C-13]G(20)[1-C-13]A(21)GGAGAGRGGLGG (II) as a local structural model of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes. Solvent treatments prior to the NMR measurements induce a structural change of these model peptides and provide a model to reproduce the structure of the silk fiber. Conformation-dependent C-13 NMR chemical shifts were mainly used to determine the local structure, including the evaluation of the fraction of several conformations. The characteristic structure, 65% beta-sheet for the Ala(8) residue in the poly-Ala region, and 70% 3(1)-helix for the Ala(21) residue and mainly 3(1)-helix for the Gly(20) residue in the GG(20)A(21) sequence was observed after peptides Ia and II were dissolved in 9 M LiBr followed by dialysis against water. The 2D spin diffusion 13C solid-state NMR spectrum of the Ala(21) residue of peptide II after this treatment was also reproduced by 70% 3(1)-helix (phi, rho = -90 degrees, 120 degrees) and 30% beta-sheet (phi, rho = -150 degrees, 150 degrees) structure. However, the Ala C-beta peak assigned to the 31-helix in the spectrum of Ia is broad, implying that the torsion angles of the Ala(21) residue are distributed, but with an average that corresponds approximately to the torsion angles of the 31-helix. An increase in the fraction of beta-sheet in both the poly-Ala and GG(20)A(21) regions was observed for la after it was dissolved in formic acid and then dried in air. Moreover, after Ia was dissolved in formic acid and then precipitated in methanol, the spectrum showed a tightly packed beta-sheet structure with a further increase in the fraction of beta-sheet although 15% 3(1)-helix still remained in the GG(20)A(21) region. The beta-sheet structure of the poly-Ala region and both 3(1)-helix and beta-sheet structures in the repeated GGA sequence are in agreement with the structural model for the native spider dragline silk fiber from N. clavipes from a previous NMR study (van Beek, J. D.; et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10266-10271). On the other hand, the a-helical conformation was found to be dominant for the peptide treated with trifluoroacetic acid together with a significant contribution from other structures. The fraction of the other structures was 20-40% depending on the position of the C-13-labeled Ala residue in the chain.